A cell-free protein synthesis system using a cell extract is utilized mainly for identification of gene products and for investigation of their activities. For example, the system enables the analysis of functions of synthesized proteins such as the enzymatic activity and DNA binding capability, or the determination of the molecular weight of translated products by labeling them with radioisotopes. Recently, techniques of drastically increasing the production amount in the system have been developed, and the system has become utilized also for protein structure analysis through X-ray crystallography, NMR and the like.
For extracts to perform the translation reaction, those derived from E. coli, wheat germ, and rabbit reticulocyte are commercially available. For an E. coli extract, S-30 cell-free extract reported by Zubay et al. (e.g., see Non-Patent Reference 1) is commonly used. For preparing the E. coli S-30 extract, RNase I-defective strains such as A19 and D10 are used. However, when the target protein is sensitive to proteolytic degradation, E. coli strain B, which is defective in ompT endoprotease and lon protease activities may be used.
For synthesizing mRNA from a cloned cDNA, the cDNA must be introduced into a suitable vector having various promoters. For increasing the protein expression efficiency, intensive promoters for phage-derived polymerases such as T7, T3, and SP6 are used at present, and various systems suitable to various types of template DNAs are commercially available. Using such cell-free systems enables cloned DNA expression in an extremely simplified manner and enables cytotoxic protein synthesis.
However, it is known that systematic and comprehensive expression of a large number of genes obtained from the recent genome analysis results in the existence of genes of relatively low expression and genes of no expression. It is believed that the low expression of genes may be caused by the reduced efficiency in the translation stage based on the difference in the nucleotide sequence as compared with the genes of higher expression.
On the other hand, it is known that antibiotics-resistant actinomycetes strains have a property of enhanced producibility of secondary metabolites (antibiotics, etc.), and it is reported that these are derived from the point mutation of ribosomal protein genes. It is suggested that, in these mutants, the conformation of the 16S ribosomal RNA (hereinafter referred to as “16SrRNA”) might change due to the point mutations of the ribosomal protein S12 and S4, which influence the mRNA reading efficiency (e.g., see Non-Patent Reference 2), but the mechanism, by which the mutation of the ribosomal protein enhances the production yield of the secondary metabolites of actinomycetes, is not yet clarified. It is reported that actinomycetes resistant to streptomycin, gentamycin and rifampicin have a property to express an extremely increased amount of a specific transcriptional regulatory protein in the growth stage (e.g., see Non-Patent Reference 3, FIG. 5). However, nothing has been clarified as yet, relating to the exogenous protein producibility in the extract of such actinomycetes cells. As compared with other bacteria, the growth speed of actinomycetes is slow and the optimum cultivation temperature thereof is low, and thus, it is difficult to provide a large quantity of cell extract of actinomycetes.
[Non-Patent Reference 1]
Geoffrey Zubay, Annual Review of Genetics, 1973, Vol. 7, pp. 267-287.
[Non-Patent Reference 2]
Yoshiko Hosoya & 3 others, Antimicrobial Agents and Chemotherapy, 1988, Vol. 42, pp. 2041-2047.
[Non-Patent Reference 3]
Haifeng Hu & 1 other, Applied and Environmental Microbiology, 2001, Vol. 67, pp. 1885-1892.